Evidence regarding the caries-inhibiting effect of chlorhexidine varnish is inconclusive. This study investigated the caries-inhibiting effect of the varnish EC40 on pits and fissures of first permanent molars. A two-year randomized controlled trial was carried out among 461 six- to seven-year-old children. In a split-mouth design, one group of molars received EC40 at baseline, 6, 12, and 18 months, and another group at baseline, 3, 12, and 15 months. Control molars did not receive EC40. Adherence to the treatment protocol was good. The dropout rate was 17%. Blinded examiners performed dental examinations.The caries-inhibiting effects of the two EC40 application schemes were comparable. The prevented fraction of caries was 25% (95%CI, 1%, 49%, p = 0.04) after 2 years and 9% (95%CI, -11%, 29%, p = 0.20) one year after termination of the trial, suggesting a short-term benefit from the use of EC40. The efficiency of EC40 is questionable in low-caries-incidence child populations.